This study explored for the first time the use of a flow-packed bed bioreactor for producing 5-acetyl-hydroxymethylfurfural (5-acetyl-HMF) and semicontinuous liquid–liquid extraction with a deep eutectic solvent (DES): choline chloride: glycerol= 1:2 (ChCl: GLY=1:2). The flow reaction system provided a specific productivity that was considerably higher than that observed in the batch reactor (1 gProduct/min·gBiocat vs. 0.07 gProduct/min·gBiocat). The biocatalyst Novozym 435® showed high operational stabilities in continuous mode and sequential batch mode without losses of activity. In continuous mode, an 80 % conversion was achieved with 100 mM 5-hydroxymethylfurfural (HMF) and a flow rate of 0.05 mL/min. The separation in the semicontinuous mode was more efficient than that for the extraction batch system (70 % vs. 38 %). Finally, in the future, a combination of continuous enzymatic transesterification and semicontinuous separation of 5-acetyl-HMF will provide an efficient, sustainable biocatalytic process that can be industrially scaled.